The calcium-sensing receptor (CaR) is a G protein-coupled, 7 transmembrane-spanning, cell surface receptor (GPCR) that binds calcium ions and allows cells to respond to changes in extracellular calcium concentrations. It plays a central role in systemic mineral metabolism by regulating parathyroid hormone secretion and renal calcium handling. In addition, it is expressed by many other cell types, including mammary epithelial cells (MECs). Expression of the CaR is low in rapidly proliferating MECs during pregnancy, but is greatly upregulated at the start of lactation. This allows the mammary gland to become a calcium-sensing organ that actively participates in the regulation of systemic calcium metabolism during lactation. In normal MECs, activation of the CaR inhibits PTHrP secretion but promotes calcium transport into milk. Therefore, if calcium delivery to the mammary gland falls, MECs transport less calcium and secrete more PTHrP. The increase in circulating PTHrP, in turn, activates bone resorption and liberates calcium from the skeleton for milk production. Thus, during lactation, a calcium-CaR-PTHrP mammary axis mimics the calcium-CaR-PTH parathyroid axis normally responsible for systemic calcium homeostasis. We have observed that malignant transformation alters the relationship between the CaR and PTHrP, such that breast cancer cells stimulate PTHrP production in response to calcium, rather than inhibiting PTHrP production, as they should. Similarly, activation of the CaR inhibits proliferation in normal cells, but stimulates proliferation in breast cancer cells. We hypothesize that these alterations in how the CaR regulates proliferation and PTHrP secretion in malignant versus normal breast cells contribute to the progression of breast tumors and the development of bone metastases. In order to investigate these possibilities, we outline three specific aims. Aim 1 will examine whether PKC phosphorylation of the CaR alters G-protein usage and cAMP and PTHrP production in response to calcium in malignant versus normal breast cells. Aim 2 will examine if disruption of the CaR gene will affect the development and progression of mammary tumors in mouse models. It will also determine if tumor CaR expression predicts outcome in a large cohort of patients with breast cancer. Aim 3 will examine if stimulation of PTHrP production by the CaR contributes to the development of osteolytic bone metastases in vivo. PUBLIC HEALTH RELEVANCE: Breast cancer is the most common neoplasm afflicting women in the US and other developed countries. This disease causes much suffering, is responsible for a large number of premature deaths and costs the US economy a great amount each year. The source of most morbidity and mortality from breast cancer is not the primary tumor itself, but rather metastases. Breast cancer is particularly prone to spread to the skeleton. It is estimated that 80% - 90% of patients with metastatic breast cancer will develop bone lesions. Bone metastases cause intractable pain, pathological fracture, hypercalcemia and spinal cord compression. They also herald mortality, as skeletal lesions cannot be cured at present. Our preliminary data suggest that the calcium sensing receptor (CaR) may contribute to the development of breast cancer and/or its bone metastases by stimulating parathyroid hormone-related protein (PTHrP) production. In contrast, during lactation, the normal breast participates in the regulation of bone metabolism by inhibiting PTHrP production. Our goal in these studies is to determine how malignant transformation alters the regulation of PTHrP production by the CaR and to determine if the CaR contributes to the development of breast cancer and/or its spread to the skeleton in animal models. It is possible that drugs developed to manipulate CaR signaling for bone and parathyroid diseases may also be effective for breast cancer.